How should I cite pysam
Pysam has not been published in print. When referring to pysam, please use the github URL: https://github.com/pysam-developers/pysam. As pysam is a wrapper around htslib and the samtools package, I suggest citing [Li.2009], [Bonfield.2021], and/or [Danecek.2021], as appropriate.
Is pysam thread-safe?
Pysam is a mix of python and C code. Instructions within python are generally made thread-safe through python’s global interpreter lock (GIL). This ensures that python data structures will always be in a consistent state.
If an external function outside python is called, the programmer has a choice to keep the GIL in place or to release it. Keeping the GIL in place will make sure that all python threads wait until the external function has completed. This is a safe option and ensures thread-safety.
Alternatively, the GIL can be released while the external function is called. This will allow other threads to run concurrently. This can be beneficial if the external function is expected to halt, for example when waiting for data to read or write. However, to achieve thread-safety, the external function needs to be implemented with thread-safety in mind. This means that there can be no shared state between threads, or if there is shared, it needs to be controlled to prevent any access conflicts.
Pysam generally uses the latter option and aims to release the GIL for I/O intensive tasks. This is generally fine, but thread-safety of all parts have not been fully tested.
A related issue is when different threads read from the same file
object - or the same thread uses two iterators over a file. There is
only a single file-position for each opened file. To prevent this from
happening, use the option
multiple_iterators=True when calling
a fetch() method. This will return an iterator on a newly opened
pysam coordinates are wrong
pysam uses 0-based coordinates and the half-open notation for ranges as does python. Coordinates and intervals reported from pysam always follow that convention.
Confusion might arise as different file formats might have different conventions. For example, the SAM format is 1-based while the BAM format is 0-based. It is important to remember that pysam will always conform to the python convention and translate to/from the file format automatically.
The only exception is the region string in the
pileup() methods. This string follows the
convention of the samtools command line utilities. The same is true
for any coordinates passed to the samtools command utilities directly,
Calling pysam.fetch() confuses existing iterators
The following code will cause unexpected behaviour:
samfile = pysam.AlignmentFile("pysam_ex1.bam", "rb") iter1 = samfile.fetch("chr1") print(next(iter1).reference_id) iter2 = samfile.fetch("chr2") print(next(iter2).reference_id) print(next(iter1).reference_id)
This will give the following output:
0 1 Traceback (most recent call last): File "xx.py", line 8, in <module> print(next(iter1).reference_id) File "libcalignmentfile.pyx", line 2103, in pysam.libcalignmentfile.IteratorRowRegion.__next__ StopIteration
Note how the second iterator stops as the file pointer has moved to chr2. The correct way to work with multiple iterators is:
samfile = pysam.AlignmentFile("pysam_ex1.bam", "rb") iter1 = samfile.fetch("chr1", multiple_iterators=True) print(next(iter1).reference_id) iter2 = samfile.fetch("chr2") print(next(iter2).reference_id) print(next(iter1).reference_id)
Here, the output is:
0 1 0
The reason for this behaviour is that every iterator needs to keep
track of its current position in the file. Within pysam, each opened
file can only keep track of one file position and hence there can only
be one iterator per file. Using the option
will return an iterator within a newly opened file. This iterator will
not interfere with existing iterators as it has its own file handle
associated with it.
Note that re-opening files incurs a performance penalty which can
become severe when calling
multiple_iterators is set to
False by default.
AlignmentFile.fetch does not show unmapped reads
fetch() will only iterate over alignments
in the SAM/BAM file. The following thus always works:
bf = pysam.AlignmentFile(fname, "rb") for r in bf.fetch(): assert not r.is_unmapped
If the SAM/BAM file contains unaligned reads, they can be included
in the iteration by adding the
bf = pysam.AlignmentFile(fname, "rb") for r in bf.fetch(until_eof=True): if r.is_unmapped: print("read is unmapped")
I can’t call AlignmentFile.fetch on a file without an index
fetch() requires an index when
iterating over a SAM/BAM file. To iterate over a file without an
bf = pysam.AlignmentFile(fname, "rb") for r in bf.fetch(until_eof=True): print(r)
BAM files with a large number of reference sequences are slow
If you have many reference sequences in a BAM file, the following might be slow:
track = pysam.AlignmentFile(fname, "rb") for aln in track.fetch(): pass
The reason is that track.fetch() will iterate through the BAM file for each reference sequence in the order as it is defined in the header. This might require a lot of jumping around in the file. To avoid this, use:
track = pysam.AlignmentFile(fname, "rb") for aln in track.fetch(until_eof=True): pass
This will iterate through reads as they appear in the file.
Weirdness with spliced reads in samfile.pileup(chr,start,end) given spliced alignments from an RNA-seq bam file
Spliced reads are reported within samfile.pileup. To ignore these
in your analysis, test the flags
is_del == True and indel == 0
I can’t edit quality scores in place
Editing reads in-place generally works, though there is one quirk to be aware of. Assigning to AlignedSegment.query_sequence will invalidate any quality scores in AlignedSegment.query_qualities. The reason is that samtools manages the memory of the sequence and quality scores together and thus requires them to always be of the same length or 0.
Thus, to in-place edit the sequence and quality scores, copies of the quality scores need to be taken. Consider trimming for example:
quals = read.query_qualities read.query_sequence = read.query_sequence[5:10] read.query_qualities = quals[5:10]
Why is there no SNPCaller class anymore?
SNP calling is highly complex and heavily parameterized. There was a danger that the pysam implementations might show different behaviour from the samtools implementation, which would have caused a lot of confusion.
The best way to use samtools SNP calling from python is to use the
pysam.mpileup() command and parse the output directly.
I get an error ‘PileupProxy accessed after iterator finished’
Pysam works by providing proxy objects to objects defined within the C-samtools package. Thus, some attention must be paid to the lifetime of objects. The following to code snippets will cause an error:
s = AlignmentFile('ex1.bam') for p in s.pileup('chr1', 1000,1010): pass for pp in p.pileups: print(pp)
The iteration has finished, thus the contents of
p are invalid. Another
variation of this:
p = next(AlignmentFile('ex1.bam').pileup('chr1', 1000, 1010)) for pp in p.pileups: print(pp)
Again, the iteration finishes as the temporary iterator created by pileup goes out of scope. The solution is to keep a handle to the iterator that remains alive:
i = AlignmentFile('ex1.bam').pileup('chr1', 1000, 1010) p = next(i) for pp in p.pileups: print(pp)
Pysam won’t compile
Compiling pysam can be tricky as there are numerous variables that differ between build environments such as OS, version, python version, and compiler. It is difficult to build software that builds cleanly on all systems and the process might fail. Please see the pysam user group for common issues.
If there is a build issue, read the generated output carefully -
generally the cause of the problem is among the first errors to be
reported. For example, you will need to have the development version
of python installed that includes the header files such as
Python.h. If that file is missing, the compiler will report
this at the very top of its error messages but will follow it
with any unknown function or variable definition it encounters later
Python 3.4 requires cython 0.20.2 or later (see here)
ImportError: cannot import name csamtools
In version 0.10.0 and onwards, all pysam extension modules contain a
lib-prefix. This facilates linking against pysam extension modules
with compilers that require to start with
lib. As a consequence,
all code using pysam extension modules directly will need to be
adapted. For example,: